In this proposal, we hypothesize that engineered thin films designed with the capacity for rapid and repeatable covalent recharging of selective molecular constituents, which block thrombin and purinergic pathways will display sustained resistance to thrombus formation in vivo. In the process, the lifetime of bioactive anti- thrombogenic films will be extended with enhanced patency of synthetic small diameter arterial substitutes. Specifically, we intend to: (1) Synthesize and characterize heterofunctional surfaces with covalently rechargeable non-fouling and anti-thrombogenic properties. In the first phase of these investigations, we plan to define the capacity of evolved mutant sortases to facilitate the fabrication of rechargeable, bioactive thin films comprised of species that block thrombin (e.g. TM, heparin) or purinergic (e.g. CD39) pathways. Synthetic heparin oligosaccharides will be used in the course of these studies; enhancing our ability to design glycocalyx-like films. The capacity of these films to block platelet thrombus formation will be determined in vitro and in vivo. During the second phase, rechargeable architectures that contain both non-fouling (e.g. polyethylene glycol) and anti-thrombogenic components will be assessed. (2) Evolve sortase-coupling systems to afford sequence specific transpeptidases with enhanced catalytic activity that facilitate in situ regeneration of biologically active surfaces. In the first phase, directed evolution will be applied to develop new orthogonal sortase variants that display sequence specificity to new peptide motifs. In order to enrich mutants for maximal activity, mutant sortase libraries cloned in yeast cells will be subjected to evolutionary pressure to enhance the affinity for LPESG, LAETG, or LAESG substrates. In the second phase, surviving sortase genes from the first phase will be diversified by gene shuffling and subjected to evolutionary pressure to further enhance catalytic activity by increasing selection stringency, reducing substrate concentration, and shortening reaction time with each successive round. (3) Determine the capacity of blood contacting devices with rechargeable anti-thrombogenic and non- fouling properties to display sustained resistance to thrombus formation in vivo. Film architectures and orthogonal sortase variants that afford rapidly rechargeable anti-thrombogenic films, which effectively inhibit platelet thrombus growth will be used to coat the luminal surface of small diameter ePTFE vascular grafts. Implant studies will be performed using a rabbit carotid interposition model to assess short- and long-term film bioactivity, stability, and rechargeability resistance to platelet deposition, as well as 30-day and 6-month patency and graft healing.